Natural human interferon β (IFN-β) is a glycoprotein consisting of 166 amino acid residues. Interferon β belong to the cytokine family, and is known to be involved in the immunoregulatory action, the anti-viral activity, and the cell growth inhibitory action. The human interferon β also has three Cys at positions 17, 31, and 141 in the natural amino acid sequence, and has one complex N-linked oligosaccharide at Asn at position 80. It is also known to have a disulfide bond at Cys at positions 31 and 141.
Interferon β as a pharmaceutical is manufactured by utilizing a cell expression system, and is classified into IFN-β-1a or IFN-β-1b depending on the difference in the host for expression. IFN-β-1a is an expression system employing Chinese hamster ovary (CHO) cells, and is a glycoprotein having sugar chains similarly to a natural interferon β. On the other hand, IFN-β-1b is expressed in E. coli, and is a protein without sugar chains.
IFN-β-1a is known to have a more potent effect compared to IFN-β-1b in regards to immunogenicity, anti-viral activity, and anti-tumor property. Further, the sugar chain structure contained in a glycoprotein is known to have a strong influence on pharmacokinetics.
It is also known that effects such as improvement in physical stability, heat stability, a plasma stability of the protein are afforded by binding a water-soluble polymer such as polyethylene glycol (PEG) to the protein. There are reports regarding PEGylated IFN-β with expectation for such effects. For example, there are reports regarding an IFN-β complex that has been PEGylated at the N-terminal of IFN-β-1b (Patent Literatures 1 and 2). There is also a report regarding an IFN-β complex that has been PEGylated at the N-terminal of IFN-β-1a (Patent Literature 3). Such modifications may in fact contribute to the above stabilities of the protein, but meanwhile causes concerns that they reduce the activity of IFN-β as a pharmaceutical. For example, it is reported that the activity is dramatically reduced in cases e.g. when the molecular weight of PEG is 20,000 or more (Non-Patent Literature 1).
With consideration for the above-described concerns of PEGylation, there is also a report of selecting a position that may maintain the activity of IFN-β even when a high molecular weight PEG for site-specific PEGylation is bound (Patent Literature 4). However, since PEG is a compound that does not exist in vivo, sufficient investigation has not yet been established in regards to the accumulatability, the safety, and the effectiveness of long-term administration of PEGylated IFN-β.
Meanwhile, there is also a report regarding site-specifically glycosylated IFN-β complexes (Patent Literature 5). In Patent Literature 5, an amino acid mutation is introduced into the amino acid sequence of natural IFNβ so that it will have a consensus sequence (Asn-X-Ser/Thr) which will be the recognition site for an N-linked sugar drain, and this is expressed with CHO cells. However, with this method, amino acid mutations other than the amino acid to be glycosylated will be produced in order to introduce the consensus sequence. It is also known in general that ununiformity of sugar chains will occur when expressed with CHO cells.